Exploring Enzyme Mechanisms and Regulation in Biochemistry

1. Enzymes: mechanisms & regulation Andy HowardIntroductory Biochemistry 17 November 2014 Enzyme Mechanisms

2. Enzyme mechanisms • Many enzymatic mechanisms involve either covalent catalysis or acid-base interactions • We’ll give some examples of several mechanistic approaches • Then we’ll talk about enzyme activity is regulated Enzyme Mechanisms

3. Mechanism Topics • Redox reactions • Induced fit • Ionic intermediates • Active-site amino acids • Serine proteases • Reaction • How they illustrate what we’ve learned • Specificity • Evolution Enzyme Mechanisms

4. Oxidation-Reduction Reactions • Commonplace in biochemistry: EC 1 • Oxidation is a loss of electrons • Reduction is the gain of electrons • In practice, often: • oxidation is decrease in # of C-H bonds; • reduction is increase in # of C-H bonds Enzyme Mechanisms

5. Redox, continued • Intermediate electron acceptors and donors are organic moieties or metals • Ultimate electron acceptor in aerobic organisms is usually dioxygen (O2) • Anaerobic organisms usually employ other electron acceptors Enzyme Mechanisms

6. Biological redox reactions • Generally 2-electron transformations • Often involve alcohols, aldehydes, ketones, carboxylic acids, C=C bonds: • R1R2CH-OH + X  R1R2C=O + XH2 • R1HC=O + X + OH- R1COO- + XH2 • X is usually NAD, NADP, FAD, FMN • A few biological redox systems involve metal ions or Fe-S complexes • Usually reduced compounds are higher-energy than the corresponding oxidized compounds Enzyme Mechanisms

7. FMN One-electron redox reactions • FMN, FAD, some metal ionscan be oxidized or reducedone electron at a time • With organic cofactors this generally leaves a free radical in each of two places • Subsequent reactions get us back to an even number of electrons Enzyme Mechanisms

8. Covalent catalysis • Reactive side-chain can be a nucleophile or an electrophile;nucleophile is more common • A—X + E X—E + A • X—E + B  B—X + E • Example: sucrose phosphorylase • Net reaction:Sucrose + Pi Glucose 1-P + fructose • Fructose=A, Glucose=X, Phosphate=B Bifidobacterium sucrose phosphorylase EC 2.4.1.7113 kDa dimer PDB 1R7A, 1.8Å Enzyme Mechanisms

9. Example: hexokinase Human brain Hexokinase IEC 2.7.1.1 104 kDa monomer PDB 1CZA, 1.9Å • Glucose + ATP  Glucose-6-P + ADP • Risk: unproductive reaction with water • Enzyme exists in open & closed forms • Glucose induces conversion to closed form; water can’t do that • Energy expended moving to closed form Enzyme Mechanisms

10. Hexokinase structure • Diagram courtesy E. Marcotte, UT Austin Enzyme Mechanisms

11. Tight binding of ionic intermediates • Quasi-stable ionic species strongly bound by ion-pair and H-bond interactions • Similar to notion that transition states are the most tightly bound species, but these are more stable Enzyme Mechanisms

12. Reactive sidechains in a.a.’s Enzyme Mechanisms

13. Generalizations about active-site amino acids • Typical enzyme has 2-6 key catalytic residues • His, asp, arg, glu, lys account for 64% • Remember: • pKa values in proteins sometimes different from those of isolated amino acids • Frequency overall  Frequency in catalysis Enzyme Mechanisms

14. Rates often depend on pH • If an amino acid that is necessary to the mechanism changes protonation state at a particular pH, then the reaction may be allowed or disallowed depending on pH • Two ionizable residues means there may be a narrow pH optimum for catalysis Enzyme Mechanisms

15. Papain as an example Enzyme Mechanisms

16. iClicker quiz, question 1 1. Why would the nonproductive hexokinase reaction H2O + ATP  ADP + Pibe considered nonproductive? • (a) Because it needlessly soaks up water • (b) Because the enzyme undergoes a wasteful conformational change • (c) Because the energy in the high-energy phosphate bond is unavailable for other purposes • (d) Because ADP is poisonous • (e) None of the above Enzyme Mechanisms

17. iClicker Quiz question 2 2. Triosephosphateisomerase (TIM) interconverts dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate. What would bind tightest in the TIM active site? • (a) DHAP (substrate) • (b) D-glyceraldehyde (product) • (c) 2-phosphoglycolate(Transition-state analog) • (d) They would all bind equally well • (e) None of them would bind at all. Enzyme Mechanisms

18. Serine protease mechanism • Only detailed mechanism that we’ll ask you to memorize • One of the first to be elucidated • Well studied structurally • Illustrates many other mechanisms • Instance of convergent and divergent evolution Enzyme Mechanisms

19. The reaction • Hydrolytic cleavage of peptide bond • Enzyme usually works on esters too • Found in eukaryotic digestive enzymes and in bacterial systems • Widely-varying substrate specificities • Some proteases are highly specific for particular amino acids at position 1, 2, -1, . . . • Others are more promiscuous O CH NH C NH C NH R1 CH O R-1 Enzyme Mechanisms

20. Mechanism • Active-site serine —OH …Without neighboring amino acids, it’s fairly unreactive • becomes powerful nucleophile because OH proton lies near unprotonated N of His • This N can abstract the hydrogen at near-neutral pH • Resulting + charge on His is stabilized by its proximity to a nearby carboxylate group on an aspartate side-chain. Enzyme Mechanisms

21. Catalytic triad • The catalytic triad of asp, his, and ser is found in an approximately linear arrangement in all the serine proteases, all the way from non-specific, secreted bacterial proteases to highly regulated and highly specific mammalian proteases. Enzyme Mechanisms

22. Diagram of first 3 steps (of 7) Enzyme Mechanisms

23. Diagram of last four steps Diagrams courtesy University of Virginia Enzyme Mechanisms

24. Chymotrypsin as example • Catalytic Ser is Ser195 • Asp is 102, His is 57 • Note symmetry of mechanism:steps read similarly L R and R  L Diagram courtesy of Anthony Serianni, University of Notre Dame Enzyme Mechanisms

25. Oxyanion hole • When his-57 accepts proton from Ser-195:it creates an R—O- ion on Ser sidechain • In reality the Ser O immediately becomes covalently bonded to substrate carbonyl carbon, moving negative charge to the carbonyl O. • Oxyanion is on the substrate's oxygen • Oxyanion stabilized by additional interaction in addition to the protonated his 57:main-chain NH group from gly 193 H-bonds to oxygen atom (or ion) from the substrate,further stabilizing the ion. Enzyme Mechanisms

26. Oxyanion hole cartoon • Cartoon courtesy Henry Jakubowski, College of St.Benedict / St.John’s University Enzyme Mechanisms

27. Modes of catalysis in serine proteases • Proximity effect: gathering of reactants in steps 1 and 4 • Acid-base catalysis at histidine in steps 2 and 4 • Covalent catalysis on serine hydroxymethyl group in steps 2-5 • So both chemical (acid-base & covalent) and binding modes (proximity & transition-state) are used in this mechanism Enzyme Mechanisms

28. What mechanistic concepts do serine proteases not illustrate? • Quaternary structural effects(We’ll discuss this under regulation…) • Protein-protein interactions(Becoming increasingly important) • Allostery(also will be discussed under regulation) • Noncompetitive inhibition Enzyme Mechanisms

29. Specificity • Active site catalytic triad is nearly invariant for eukaryotic serine proteases • Remainder of cavity where reaction occurs varies significantly from protease to protease. • In chymotrypsin  hydrophobic pocket just upstream of the position where scissile bond sits • This accommodates large hydrophobic side chain like that of phe, and doesn’t comfortably accommodate hydrophilic or small side chain. • Thus specificity is conferred by the shape and electrostatic character of the site. Enzyme Mechanisms

30. Chymotrypsin active site • Comfortably accommodates aromatics at S1 site • Differs from other mammalian serine proteases in specificity Diagram courtesy chemistry program, Eastern Washington Univ. Enzyme Mechanisms

31. Divergent evolution • Ancestral eukaryotic serine proteases presumably have differentiated into forms with different side-chain specificities • Chymotrypsin is substantially conserved within eukaryotes, but is distinctly different from elastase Enzyme Mechanisms

32. iClicker quiz, question 3 3. Why are proteases often synthesized as zymogens? • (a) Because the transcriptional machinery cannot function otherwise • (b) To prevent the enzyme from cleaving peptide bonds outside of its intended realm • (c) To exert control over the proteolytic reaction • (d) None of the above Enzyme Mechanisms

33. iClicker question 4 4. Which of these enzymes would you predict to be the most similar to human pancreatic elastase? • (a) human pancreatic chymotrypsin • (b) porcine pancreatic elastase • (c) subtilisin from Bacillus subtilis • (d) none of these would be very similar to human pancreatic elastase Enzyme Mechanisms

34. Convergent evolution • Reappearance of ser-his-asp triad in unrelated settings • Subtilisin: externals very different from mammalian serine proteases; triad same Enzyme Mechanisms

35. Subtilisin mutagenesis • Substitutions for any of the amino acids in the catalytic triad has disastrous effects on the catalytic activity, as measured by kcat. • Km affected only slightly, since the structure of the binding pocket is not altered very much by conservative mutations. • An interesting (and somewhat non-intuitive) result is that even these "broken" enzymes still catalyze the hydrolysis of some test substrates at much higher rates than buffer alone would provide. I would encourage you to think about why that might be true. Enzyme Mechanisms